The present invention is directed to a process for isolating living target cells from biological tissue.
Cells which are strongly interconnected to form a biological tissue like epithelial cells are difficult to isolate into single, living cells from the tissue. While it is possible to mechanically destroy the infrastructure of the biological tissue and isolate single cells from the resulting debris, the thus obtained yield of living, unharmed cells is rather low.
It is known to isolate cells from organs in a more gentle perfusion process, but this requires cumbersome perfusion of the organ through an appropriate blood vessel with a sequence of buffer solutions. Such processes are known for the isolation of cardiomyocytes, or hepatocytes etc.
The procedure to isolate intact hepatocytes was first introduced by Howard and Pesch (1967) J Cell Biol 35, 675-684 and refined by Berry and Friend (1969) J Cell Biol 43, 506-520; Seglen (1976) Methods Cell Biol. 13, 29-34 and Klaunig (1981) In Vitro 17, 913-925. The outcome was a two-stage perfusion protocol which, after decades, still represents the current standard procedure leading to high amounts of viable hepatocytes. Usually, a loose ligature is placed around the appropriate blood vessel (rat: vena portae; mouse: vena cava) near the liver, a cannula is inserted into the blood vessel, and the ligature is fixed by tightening the thread ends. First, a calcium depleting buffer containing EDTA or EGTA is delivered into the organ through the blood vessel at a flow rate of about 10-15 ml/min for 10-15 min in order to weaken calcium-depending cell-cell junctions and remove blood cells. Second, a buffer comprising collagenase (at about 0.1 Wünsch Units per ml) is channeled (rat: 10-15 min; mouse: 5-10 min) through the organ at a flow rate of about 10-15 ml/min for destruction of connective tissue further liberating cells without damaging target cells. The liver can now be removed from the animal and placed in an appropriate media on a petri dish. Tearing apart the liver capsule leads to the release of liberated hepatocytes which are further purified by filtration through a 100 μm nylon mesh filter and an (optionally repeated) low-spin centrifugation step (5 min 50 g at 4° C.) to enrich hepatocytes.
A similar but ex-vivo process is used for hepatocyte isolation from livers of bigger vertebrates and/or for the isolation of adult cardiomyocytes. In these applications, the target organ is excised before cannulating a blood vessel.
For the generation of human hepatocytes see review of Gomez-Lechon and Castell, “Isolation and culture of human hepatocytes” in Berry and Edwards, The Hepatocyte Review, (2000), 11-15. The tissue is put on ice as fast as possible and inspected for large vessels. Cannulae are inserted into the largest vessels of the cut surface and fixed with tissue glue. In a first step, the liver tissue is perfused extensively with an calcium-depleting buffer containing EGTA (or EDTA) at about 10 ml/catheter. In the second step, it is perfused at the same flow rate with an about 0.2 U/ml collagenase-containing enzyme mix until the tissue shows irreversible deformation. Mostly, the enzyme buffer is recirculated to reduce costs. After digestion, the liver tissue is gently dispersed with a spatula. Analogues to the rodent procedure, the cell suspension is filtered and hepatocytes are enriched by a (optionally repeated) low-spin centrifugation.
Cardiomyocytes are generated by the so-called Langendorff perfusion system. The heart is rapidly excised from the body and, under a microscope, a cannula is carefully inserted into the aorta and fixed with a ligature. Then, the heart is perfused with a calcium-free buffer to arrest contraction, followed by a collagenase-based enzyme solution to digest the extracellular matrix of the heart. After digestion, the heart is mechanically dissociated with forceps and cardiomyocytes are dispersed into a single-cell suspension.
Lung alveolar epithelial cells are isolated in a similar procedure by inserting and fixing a cannula in the trachea and applying appropriate buffer and enzymes.
US20110295149 discloses a device to solubilize tissue by an abrasive extraction of tissue fragments. The device is fixed on the tissue by vacuum and the cells are cut from the tissue with an abrasive component and further liquefied by appropriate enzymes.
In summary, the known isolation processes for cells from biological tissue are elaborate, time-consuming and are always executed by skilled personal.
In another technical field, it is known to inject pharmacological active compounds into tissue like skin through a plurality of cannulas. The cannulas can be assembled in arrays having a common input lumen, as for example disclosed in U.S. Pat. Nos. 6,689,103, 8,349,554B2, 8,366,677B2, 8,708,965B2, US2011/0213335A1, WO2014/047287A1 or EP2749306. These publications are silent on the dissociation of the tissue to generate single-cells.